p-Coumaric Acid and Ursolic Acid from Corni fructus Attenuated β

May 12, 2014 - Pheochromocytoma. PC12 cells (ATCC CRL 1721) have been extensively used as an in vitro model system to study the mechanisms of ...
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p‑Coumaric Acid and Ursolic Acid from Corni fructus Attenuated β‑Amyloid25−35-Induced Toxicity through Regulation of the NF-κB Signaling Pathway in PC12 Cells Jeong-Hyun Yoon,†,∥ Kumju Youn,†,∥ Chi-Tang Ho,‡ Mukund V. Karwe,‡ Woo-Sik Jeong,§ and Mira Jun*,† †

Department of Food Science and Nutrition, Dong-A University, Busan 604-714, Korea Department of Food Science, Rutgers University, New Brunswick, New Jersey 08901, United States § Department of Food and Life Science, College of Biomedical Science and Engineering, Inje University, Gimhae 621-749, Korea ‡

ABSTRACT: Neuroinflammatory responses induced by amyloid-beta peptide (Aβ) are important causes in the pathogenesis of Alzheimer’s disease (AD). Blockade of Aβ has emerged as a possible therapeutic approach to control the onset of AD. This study investigated the neuroprotective effects and molecular mechanisms of p-coumaric acid (p-CA) and ursolic acid (UA) from Corni fructus against Aβ25−35-induced toxicity in PC12 cells. p-CA and UA significantly inhibited the expression of iNOS and COX-2 in Aβ25−35-injured PC12 cells. Blockade of nuclear translocation of the p65 subunit of nuclear factor κB (NF-κB) and phosphorylation of IκB-α was also observed after p-CA and UA treatment. For the upstream kinases, UA exclusively reduced ERK1/2, p-38, and JNK phosphorylation, but p-CA suppressed ERK1/2 and JNK phosphorylation. Both compounds comprehensively inhibited NF-κB activity, but possibly with different upstream pathways. The results provide new insight into the pharmacological modes of p-CA and UA and their potential therapeutic application to AD. KEYWORDS: Alzheimer’s disease, amyloid β peptide, Corni fructus, p-coumaric acid, ursolic acid, COX-2, iNOS, NFκB, IκB, MAPKs



resulting free NF-κB then binds to κB binding sites in the promoter regions of target genes that regulate transcription of pro-inflammatory mediators.8 Activation of the NF-κB signaling pathway is intimately related with the activations of mitogenactivated protein kinase (MAPKs), which further stimulate downstream transcription factors that promote pro-inflammatory gene expression.9 Characteristic MAPKs subfamily members are extracellular signal regulated kinase 1/2 (ERK 1/2), c-Jun NH2-protein kinase (JNK), and p38.10−12 These activated MAPKs phosphorylate and activate other kinases or transcription factors, consequently resulting in the alteration of the target gene expression.13 Corni fructus (CF), the fruit of Cornus officinalis Sieb. et Zucc, is a well-known food and medicinal herb in China, Japan, and Korea. CF has been shown to possess antidiabetic, antiinflammatory, and antioxidative properties.14,15 In our previous study, p-coumaric acid (p-CA) and ursolic acid (UA) were isolated from the ethyl acetate fraction of CF ethanol extract. We found that p-CA and UA specifically inhibited BACE116 and further suppressed Aβ25−35-induced reactive oxygen species (ROS) generation and apoptotic activity in PC12 cells.17 To extend our knowledge of the neuroprotective effects of p-CA and UA, the anti-inflammatory activity of p-CA and UA against Aβ25−35-induced injury in PC12 cells was investigated and their underlying molecular mechanism of neuroprotective action was elucidated for the first time.

INTRODUCTION Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by progressive loss of neurons in the brain, which correlates with deposition of neurofibrillary tangles and accumulation of senile plaques, the two pathological hallmarks of the disease.1 β-Amyloid (Aβ) peptide, a 40−42 residue of the internal peptide segment from amyloid precursor protein (APP), is the major component of senile plaques and has been considered to play a pivotal role in the development and progress of AD.2 Aβ is liberated by the sequential actions of two proteases, βsecretase (BACE1) and γ-secretase. There is compelling evidence indicating that extracellular deposits of Aβ may induce activation of astrocytes and microglial cells by releasing pro-inflammatory factors that contribute to the neurodegeneration process in AD.3 Various epidemiological studies have proved that the use of conventional NSAIDs can prevent and/ or retard AD, which supported the notion that the neuroinflammatory responses can be an attractive target for treatment with anti-inflammatory drugs.4 NF-κB is a transcription factor that plays a key role in the regulation of genes associated with inflammation, and its activation is one of the signaling pathways through which Aβ exerts its neurotoxicity.5 The activated NF-κB regulates various pro-inflammatory mediators such as inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and pro-inflammatory cytokines such as interleukins (ILs) and tumor necrosis factor α (TNFα).6,7 When unstimulated, NF-κB exists mainly as inactive heterodimers of p50/p65 in the cytoplasm binding with an inhibitory protein, IκB. On the other hand, in response to cellular stimulation, the IκB kinase complex is degraded and the © 2014 American Chemical Society

Received: Revised: Accepted: Published: 4911

March 17, 2014 May 10, 2014 May 11, 2014 May 12, 2014 dx.doi.org/10.1021/jf501314g | J. Agric. Food Chem. 2014, 62, 4911−4916

Journal of Agricultural and Food Chemistry



Article

MATERIALS AND METHODS

General. Aβ25−35, p-CA, and UA were purchased from Sigma Chemical Co. (St. Louis, MO, USA). RPMI 1640 medium, phosphatebuffered saline (PBS), fetal bovine serum, donor equine serum, trypsin 0.25% solution, and penicillin/streptomycin solution were obtained from Hyclone Laboratories, Inc. (Logan, UT, USA). N2 supplement and RPMI 1640 phenol red free medium were purchased Gibco BRL (Grand Island, NY, USA). Rabbit-antiphospho-iNOS, goat-antiphospho-COX-2, and goat-anti-β-actin were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Rabbit antiphospho-JNK, rabbit-antiphospho-ERK, rabbit-antiphospho-IκB-α, and rabbit-antiphospho-p65 monoclonal antibodies were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA). All other reagents used in this study were of the highest grade available. Peptide Preparation and Cell Culture. Pheochromocytoma PC12 cells (ATCC CRL 1721) have been extensively used as an in vitro model system to study the mechanisms of neuronal toxicity.18,19 PC12 cells were purchased from ATCC (Manassas, VA, USA). PC12 cells were maintained in RPMI 1640 supplemented with 10% heatinactivated horse serum and 5% fetal bovine serum at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. All cells were cultured in serum-free medium for 16 h prior to sample treatment. Samples were added at indicated concentrations 1 h prior to Aβ25−35 treatment, and the cells were plated at a density of 2 × 106 cells/well. The Aβ25−35 peptide used in this study preserves the toxicity of the full length Aβ and is widely used to model Aβ-induced neurodegeneration. The fragment Aβ25−35 has the central functional domain of the whole molecules Aβ that is required for neurotoxic effect.20,21 Aβ25−35 was dissolved in DMSO at a concentration of 10 mM and stored at −20 °C. The stock solution was diluted with PBS to appropriate concentration and incubated for 48 h at 37 °C for aggregation prior to each experiment. Samples were dissolved in DMSO and further diluted with PBS. The final concentration of DMSO was adjusted to